Many biological processes are regulated by proteins. Regulatory proteins undergo conformational changes to alter their interactions with partners and/or alter their catalytic efficiency. Thus, it is essential to detect conformational changes of proteins in order to understand the molecular mechanism underlying their functions. Although a large body of in vitro studies has revealed conformational changes of proteins, there are no established techniques to monitor protein conformational changes in the cellular environment. Biophysical measurements, such as X-ray crystallography, nuclear magnetic resonance, and other spectroscopies, typically require purified samples and conditions that are drastically different from those inside the cells. It is generally accepted that the “molecular crowding” within the cellular environment can significantly affect ligand binding, catalysis, stability and folding of macromolecules (Minton, 2000). For example, the structures and the relative populations of “active” and “inactive” conformations of a protein may be quite different from those determined using in vitro biophysical methods. Therefore, it would be of great value to establish a strategy to probe conformations of proteins in living cells.
An alternative approach to direct structure determination is the use of conformation-specific probes. Anfinsen and others used conformation-specific antibodies to demonstrate reversible unfolding of ribonuclease in in vitro experiments (Sachs et al., 1972). Thus, it is conceivable that one can introduce conformation-specific probes, such as antibodies, inside cells and determine their respective binding affinity to a target to probe conformational changes of the target. To implement this strategy, one must first obtain conformation-specific probes and establish detection methods for probe binding. However, antibodies and their fragments usually require the formation of disulfide bonds for proper folding and, thus, they do not always function in the reducing environment inside cells. Also, no general methods are available to generate conformation-specific antibodies. Short peptides may also be used, but they tend to be rapidly degraded in cells due to their low resistance to proteolysis.
Antibody-mimics, termed “monobodies”, formed using a small β-sheet protein scaffold such as the tenth fibronectin type m domain from human fibronectin (FNfn10) have been previously described (Koide et al., 1998). It was shown that monobodies with a novel binding function can be engineered by screening phage-display libraries of FNfn10 in which loop regions are diversified. FNfn10 does not contain disulfide bonds or metal binding sites, is highly stable and undergoes reversible unfolding (Koide et al., 1998; Main et al., 1992; Plaxco et al., 1996). While the stability of monobodies makes them well suited for intracellular studies, there has been no use of monobodies to probe conformations of proteins in living cells.
A number of disease states are dependent upon nuclear receptor activity and conformation. For example, human estrogen receptor α (ERα) normally regulates the growth and differentiation of the female reproductive system and those of skeletal, neural, and cardiovascular tissues in both males and females (Korach, 1994). Yet ERα is a therapeutic target of, and a clinical marker for, estrogen-responsive breast tumor (Jordan et al., 1992). A diverse group of ligands, including antiestrogens that are in clinical use, exist which modulate ER transcriptional activation and the physiological response of the hormone 17β-estradiol (E2) (Anstead et al., 1997). Because the conformation of ERα as it is involved in disease state is unknown, it would be desirable to identify an approach to rapidly classify ERα conformation as well as develop a preliminary screening tool for estrogen- and antiestrogen-like molecules. Any approach which would function to classify ERα conformation and screen estrogen- and antiestrogen-like molecules should also be able to be operable with other nuclear receptors: classifying their conformations and screening their agonists and antagonists.
In addition to screening, another important feature in drug discovery is target validation. The majority of target validation methods are based on nucleic acid techniques. These include gene knockout (the gene coding for the protein of interest is eliminated from the genome of the organism) and antisense DNA (DNA that hybridize to the messenger RNA of the protein of interest is produced in the cell to inhibit the expression of the protein). These techniques are limited in that some genes are essential for the growth of the organism and cannot be deleted, and the effect of deleting a protein may be different from inhibiting its function (sometimes only partially) with drugs.
Recently, however, a few methods based on protein technologies have been reported (Mhashilkar et al., 1995; Richardson et al., 1995; Colas et al., 1996; Cochet et al., 1998; Colas & Brent, 1998; Fabbrizio et al., 1999; Norris et al., 1999). Proteins or peptides that bind to the protein of interest (“peptide aptamers”) are first isolated (typically using combinatorial library screening). Then the peptide aptamer is introduced into the organism of interest (typically using an expression vector), and the effect(s) of the aptamer is analyzed. For peptide aptamers, constrained peptides that are displayed on a protein (Colas et al., 1996; Fabbrizio et al., 1999), linear peptides (Norris et al., 1999), and antibody fragments (Mhashilkar et al., 1995) have been reported. Though these approaches have been at least in some sense successful, they have their limitations. The first two methods use only one contiguous segment of peptides for binding, and thus the binding interface achieved by these methods is limited. Antibody fragments (e.g, single-chain Fv and Fab) contain disulfide bonds, and these disulfide bonds are important for the stability of antibody fragments. The cytoplasm of the cell is generally a reducing environment, making it difficult to maintain the active conformation of antibody fragments. Thus, antibody fragments expressed in the cytoplasm are not always functional (Cochet et al., 1998).
The present invention overcomes these and other deficiencies in the art.